Reduction and denaturation before affinity chromatography for host cell protein removal in aggregate-rich monoclonal...

Host cell proteins (HCPs) are important process-related safety concern factors during drug development, clinical trials and manufacturing, and their removal becomes particularly challenging when associated with antibody aggregates. Residual HCPs in drug products could trigger adverse effects or compromise drug efficacy and stability. This study aims to enhance the HCP removal specifically in monoclonal antibodies (mAbs) with high aggregate content before protein A chromatography by introducing reduction and denaturation steps. A model mAb was purified by different processes, and the impact on HCP removal was evaluated by enzyme-linked immunosorbent assay (ELISA) and Liquid chromatography-mass spectrometry (LC-MS). The HCP profiles in high aggregate, low aggregate and monomeric mAbs were analyzed by LC-MS. The additional reduction and denaturation steps improved HCP removal rate by 1.08 log10 reduction value (LRV) compared to the control process. For the relatively abundant high-risk HCPs, 45% were completely removed. HCP removal in the model mAb was significantly enhanced, especially in high aggregate forms. This method can reduce HCP carry-over from mAbs aggregates in subsequent downstream processing and can provide an efficient HCP removal strategy for manufacturing to improve patient safety and drug stability.